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c-Myc-tag control-lysate
Description
The c-Myc-tag control-lysate was generated from
transfected HEK293 cells, expressing a c-Myc
tagged protein. The c-Myc-tag corresponds to
a decapeptide (EQKLISEEDL) of the c-Myc proto-oncoprotein and belongs to the most widely
used epitope-tags.
To generate the c-Myc-tag control-lysate HEK293
cells were transiently transfected with a mammalian
expression vector coding for a cytoplasmic
protein harbouring an amino-terminal c-Myc-tag.
A whole cell lysate was prepared from these transiently
transfected cells. Besides all other cellular proteins,
this whole cell lysate contains the c-Myc-tag
protein, which shows an apparent molecular
weight of ~21 kDa upon SDS-PAGE.
In addition to the c-Myc-tag control-lysate, a
mock-lysate was prepared from HEK293 cells,
which were transfected with the empty
expression vector.
Product
Whole cell lysate:
50 µl
Cell line:
HEK293 cells
Tagged protein:
Human cytoplasmic protein
with an amino-terminal c-Myc-tag.
MW:
~21 kDa
Lysis buffer:
60 mM Tris/HCL pH 6.8, 2% SDS,
5% mercaptoethanol,
50 µg/ml bromophenolblue, 10% glycerol
Application:
For SDS-PAGE/Western Blotting
20 µl of the lysates should be applied per
lane of a 15% polyacrylamide-gel.
Shipping and storage
Shipping:
The lysate is shipped in cold case.
Storage:
Lysate is stable for 1 week at 4°C.
For prolonged storage, the lysate should be stored
at -80°C. Aliquot to avoid repeated freezing and thawing.
At -80°C, the product is stable for at least 1 year
from shipment.
Application in quality control
Western Blotting using the c-Myc-tag control-lysate and the mock-lysate.
HEK293 cells were transiently transfected either
with the empty expression vector (mock-lysate;
mock-lys.) or with an expression vector
encompassing the cDNA of a c-Myc-tag protein
(c-Myc-tag control-lysate). 25 µl of the mock-lysate
and the indicated amounts of the c-Myc-tag control-lysate (25, 20, 15, 10, or 0 µl) were separated by
SDS-PAGE using a 15% polyacrylamide-gel and
then transfered onto a PVDF membrane. The
membrane was probed with the monoclonal
anti-c-Myc-tag antibody from tag-tools (clone TT5; dilution 1:2,000).
The c-Myc-tag bound primary antibody was
detected by incubation with HRP-coupled
protein G (dilution 1:10,000) and visualized using
chemiluminescence.
The X-ray film was exposed for 30 seconds.
Whereas a specific ~21 kDa band is detected
in the c-Myc-tag control-lysate at all volumes
(10 - 25 µl), no binding of the anti-c-Myc-tag
antibody to cellular proteins can be detected
in the lane, where 25 µl of the mock-lysate
was applied.
Use
The c-Myc-tag control-lysate and the mock-lysate
are for quality control in research applications only.
Not for diagnostic or therapeutic purpose.
